Regulation of IL-8 by Irsogladine maleate is involved in abolishment of Actinobacillus actinomycetemcomitans-induced reduction of gap-junctional intercellular communication

Our previous report has shown that Irsogladine maleate (IM) counters and obviates the reduction in gap junction intercellular communication (GJIC) and the increase in IL-8 levels, respectively, induced by outer membrane protein 29 from Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans) in cultured human gingival epithelial cells (HGEC). In addition, IM suppresses the increase in the secretion of IL-8 caused by whole live A. actinomycetemcomitans. These findings implicate the modulation of IL-8 levels by IM in abolishment of the reduction of GJIC in HGEC. Tight junctions are also responsible for cell-cell communication. Zonula occludens protein-1 (ZO-1) is a major tight junction protein. To investigate the regulatory mechanism of intercellular communication mediated by IM, in the present study, we focused on the involvement of IL-8 in A. actinomycetemcomitans-induced change in GJIC and ZO-1 expression in HGEC. IM countered the A. actinomycetemcomitans-induced reduction in levels of Connexin (CX) 43, suggesting that it could abolish the A. actinomycetemcomitans-induced reduction in GJIC in HGEC. CXCR-1 is a receptor of IL-8. The simultaneous addition of A. actinomycetemcomitans and anti-CXCR-1 antibody also abrogated the repression of GJIC and CX43 expression by A. actinomycetemcomitans in HGEC, although the anti-CXCR-1 antibody was less effective than IM. IM inhibited the IL-8-induced reduction in CX43 levels and GJIC in HGEC. IM countered the A. actinomycetemcomitans-induced reduction in the expression of ZO-1, although anti-CXCR-1 antibody did not influence the decrease in ZO-1 mRNA levels caused by A. actinomycetemcomitans. Furthermore, IL-8 had little effect on the mRNA levels of ZO-1. These findings suggest that IL-8 mediates the A. actinomycetemcomitans-induced reduction of GJIC and CX43 expression in HGEC. The regulation of IL-8 levels by IM in HGEC is partially involved in abrogation of the reduction of GJIC and CX43 expression by A. actinomycetemcomitans. Furthermore, the regulatory effect of IM on the expression of CX43 and ZO-1 is different.     

IL-15 up-regulates iNOS expression and NO production by gingival epithelial cells

To investigate the biological activity of epithelial cells in view of host defense, we analyzed the mRNA expression of inducible NOS (iNOS) as well as NO production by human gingival epithelial cells (HGEC) stimulated with IL-15. RT-PCR analysis revealed that HGEC expressed IL-15 receptor alpha-chain mRNA. In addition, stimulation with IL-15 enhanced iNOS expression by HGEC through an increase of both mRNA and protein levels. Moreover, IL-15 up-regulated the production of NO(2)(-)/NO(3)(-), a NO-derived stable end product, from HGEC. The enhanced NO production by IL-15 was inhibited by AMT, an iNOS-specific inhibitor. These results suggest that IL-15 is a potent regulator of iNOS expression by HGEC and involved in innate immunity in the mucosal epithelium.     

Hepatocyte growth factor and keratinocyte growth factor enhance IL-1-induced IL-8 secretion through different mechanisms in Caco-2 epithelial cells

A variety of cytokines have been detected in inflamed intestinal mucosal tissues, including the pro-inflammatory cytokine, interleukin-1 (IL-1), along with growth factors involved in wound healing processes such as proliferation and cell migration. However, little is known about how IL-1 and growth factors interact with intestinal epithelial cells to regulate the production of inflammatory cytokines such as interleukin-8 (IL-8). Previously, we have shown that hepatocyte growth factor (HGF) could significantly enhance IL-1-stimulated IL-8 secretion by the Caco-2 colonic epithelial cell line, yet HGF, by itself, did not stimulate IL-8 secretion. In this report, a second growth factor, keratinocyte growth factor (KGF), was also found to significantly enhance IL-1-induced IL-8 secretion by Caco-2 cells, yet KGF, by itself, also had no effect. Simultaneous addition of both IL-1 and KGF was also required for the enhancing effect. Treatment of the Caco-2 cells with wortmannin or triciribine suppressed the enhancing effect of HGF, suggesting that the effect was mediated by signaling through phosphatidylinositol-3-kinase (PI3K) and the kinase AKT. The enhancing effect of KGF was not affected by wortmannin, but was suppressed by triciribine, suggesting that the effect of KGF was through a PI3K-independent activation of AKT. These results suggest that the growth factors HGF and KGF may play a role in enhancing IL-1-stimulated production of IL-8 by epithelial cells during mucosal inflammations. However, the mechanism by which the growth factors enhance the IL-1 response may be through different initial signaling pathways.     

IL-1 stimulates IL-6 production in endothelial cells

Leukocytes and vascular cells interact closely in inflammation and immunity and lymphokines are important mediators of this interaction. The present study was designed to define the possible role of IL-6 as a communication signal between vascular and immunocompetent cells. IL-6 was measured as hybridoma growth factor (HGF) on the 7TD1 cell line in the supernatants of human endothelial cells (HEC). HEC released appreciable levels of HGF activity in the absence of deliberate stimulation. In vitro exposure to recombinant IL-1 beta markedly increased (usually 10 to 15-fold) HGF production by HEC. Optimal stimulation was observed with 0.1 to 50 U/ml for 4 to 20 h of incubation. Human and murine rIL-1 alpha stimulated HGF production in HEC. Anti-IL-6 antibodies inhibited the HGF activity of the HEC supernatants, thus confirming, together with the cytokine specificity of the assay, the nature of HEC-produced cytokine. IL-1-treated HEC expressed high levels of IL-6 mRNA as detected by Northern blot analysis. Inasmuch as IL-1 elicits a complex series of changes in HEC, it was important to assess whether IL-6, produced after exposure to IL-1, modified HEC function. Natural or rIL-6 did not affect the functional status of HEC as assessed by proliferative capacity, production of procoagulant activity and prostacyclin, ability to induce adhesion of polymorphonuclear leukocytes. The capacity to produce IL-6 may represent an important mechanism by which endothelial cells participate in inflammatory and immune reactions.     

Actinobacillus actinomycetemcomitans possesses an antigen binding to anti-human IL-10 antibody

It is well known that the cell components of periodontopathic bacteria are able to induce several cytokines and possibly to affect the cytokine network. In order to determine the presence of the periodontopathic Actinobacillus actinomycetemcomitans components recognized by antibodies against cytokine molecules, ELISA reactivities of sonic extracts from the bacterial cells were determined by use of ELISA kits specific for human interleukin (IL)-1beta, IL-4, IL-5, IL-6, IL-10, tumor necrosis factor-alpha, and interferon-gamma. The ELISA analysis demonstrated that the sonic extracts from eight strains of A. actinomycetemcomitans bound with anti-human IL-10 monoclonal antibody. Western blotting analysis revealed that the molecular mass of the antigen was approximately 65 kDa. IL-10 is produced by type 2 helper T cells and mainly down-regulates the type 1 helper T cell response. The present study suggests that the 65-kDa antigen of A. actinomycetemcomitans may affect the host defense function through binding to IL-10 receptor as an agonist or an antagonist for IL-10.     

Hepatocyte growth factor enhances IL-1β stimulated IL-8 secretion by Caco-2 epithelial cells

Hepatocyte growth factor (HGF) can induce proliferation and migration of intestinal epithelial cells and has also been shown to be important in wound healing of inflamed mucosal tissues. HGF is known to be expressed along with interleukin-1 (IL-1) by inflamed mucosal tissues, yet the effect of HGF on IL-1-induced proinflammatory cytokine responses by colonic epithelial cells is unknown. In this report, we have examined the effect of HGF on IL-1-induced secretion of IL-8 by the Caco-2 colonic epithelial cell line. HGF stimulation alone had no effect on the secretion of IL-8 by the Caco-2 cells. However, culture of the cells with HGF and suboptimal levels of IL-1 resulted in a significant enhancement of IL-8 secretion compared to cells cultured with IL-1 alone. A similar effect was seen with HGF and IL-1 simulation of monocyte chemoattractant protein-1 secretion by the rat IEC-6 intestinal epithelial cell line. The enhancing effect of HGF was seen regardless of whether the culture medium contained serum or not. Simultaneous stimulation with HGF and IL-1 was required for the enhancing effect as cells pretreated with HGF for 24 h and then stimulated with IL-1 alone secreted IL-8 levels similar to that of cells stimulated with IL-1 alone. These results suggest that in addition to wound healing, HGF may play a role in the IL-1-induced chemokine response of epithelial cells in inflamed mucosal tissues.     

Up-regulation of hepatocyte growth factor gene expression by interleukin-1 in human skin fibroblasts.

Hepatocyte growth factor (HGF) functions as a hepatotrophic and renotrophic factor for regeneration of the liver and kidney. When 1 ng/ml of interleukin-1 alpha (IL-1 alpha) or interleukin-1 beta (IL-1 beta) was added to cultures of human skin fibroblasts, the production of HGF was 5-6 fold higher than levels in the controls. HGF mRNA level in the cells was increased to 4-fold higher levels at 6 h after exposure to IL-1 alpha. Tumor necrosis factor-alpha and interferon-gamma but no other cytokine tested had slightly stimulatory effects on HGF production. The tumor promoter, tetradecanoylphorbol 13-acetate (TPA) markedly enhanced the stimulatory effect of IL-1 alpha and IL-1 beta on the production of HGF. The stimulatory effect of both IL-1 alpha and IL-1 beta and the synergistical stimulation with TPA were completely abrogated by 10 ng/ml TGF-beta 1 or 1 microM dexamethasone. These results suggest that IL-1 alpha and IL-1 beta are positive regulators for expression of the HGF gene and are likely have a role in regeneration of tissues following the occurrence of inflammatory diseases.     

IL-1beta-mediated proinflammatory responses are inhibited by estradiol via down-regulation of IL-1 receptor type I in uterine epithelial cells

The objective of this study was to examine the effects of sex hormones on IL-1beta-mediated responses by uterine epithelial cells. The mRNA expression and secretion of human beta-defensin-2 and CXCL8 by uterine epithelial cells was examined following stimulation with IL-1beta in the presence of estradiol or progesterone. Estradiol inhibited the IL-1beta-mediated mRNA expression and secretion of human beta-defensin-2 and CXCL8 by uterine epithelial cells while progesterone had no effect. Inhibition of the IL-1beta-mediated response by estradiol was dose dependent, with maximal inhibition observed using 10(-7) to 10(-10) M, and was shown to be mediated through the estrogen receptor because addition of a pure estrogen receptor antagonist abrogated this effect. The mechanism by which estradiol inhibits IL-1beta-mediated responses by uterine epithelial cells appears to be the down-modulation of the IL-1R type I, thereby reducing the uterine epithelial cell's ability to respond to IL-1beta. These results suggest that the inhibitory effect of estradiol on IL-1beta-mediated inflammatory responses by uterine epithelial cells indicates a link between the endocrine and immune systems and may be crucial for dampening proinflammatory responses during the time of ovulation or pregnancy.     

Endogenous IL-15 sustains recruitment of IL-2Rbeta and common gamma and IL-2-mediated chemokine production in normal and inflamed human gingival fibroblasts

We recently reported that anti-IL-15 neutralizing mAb has been shown to inhibit production of MCP-1 in response to IL-2 from normal human gingival fibroblasts (HGF), the major constituent of gingival tissue. In the present study, we examined the expression of IL-2R and IL-15R subunits in HGF from normal and inflamed regions and the role of endogenous IL-15 in IL-2-mediated signaling. Normal HGF expressed IL-2Rbeta and common gamma-chain (gammac) but not IL-2Ralpha or IL-15Ralpha, whereas inflamed HGF expressed IL-2Ralpha, IL-15Ralpha, IL-2Rbeta, and gammac, as assessed by RT-PCR and flow cytometry. Exogenous IL-2 and IL-15 induced production of MCP-1 but not IL-8 in normal HGF, and induced the production of both chemokines in inflamed HGF. Both HGF constitutively transcribed the 48 aa-IL-15 isoform, and the isoform was not actively secreted but rather existed as a membrane-bound form. Pretreatment with anti-IL-15 neutralizing mAb for 24 h completely inhibited the production of MCP-1 induced by IL-2 and IL-15 and IL-2-induced phosphorylation of Jak 1 and 3 in HGF. The pretreatment and RNA interference targeted to IL-15 mRNA resulted in total inhibition of the IL-2Rbeta and gammac expression at mRNA and protein levels. Furthermore, excess amounts of IL-2 restored the inhibitory effect of anti-IL-15, inhibition of NF-kappaB abrogated the expression of IL-2Rbeta and gammac, and IL-2-induced-nuclear translocation of NF-kappaB was completely inhibited by the RNA interference in HGF. These results suggest that endogenous membrane-bound IL-15 sustains recruitment of IL-2Rbeta and gammac through activation of NF-kappaB in HGF.     

Anti-inflammatory effects of hepatocyte growth factor: induction of interleukin-1 receptor antagonist

Hepatocyte growth factor (HGF) prevents liver failure in various animal models including endotoxin-induced acute liver failure. We were interested to find out whether human HGF exerts anti-inflammatory effects by modulation of cytokine synthesis. Therefore, human HepG2 cells were cultured with increasing concentrations of HGF. HGF dose-dependently upregulated the production of interleukin-1 receptor antagonist (IL-1Ra). Incubation of HepG2 cells with interleukin-1beta (IL-1beta) caused an increase in IL-1Ra levels, while interleukin-6 (IL-6) had no effect on IL-1Ra synthesis. Co-stimulation of HepG2 cells with HGF + IL-1beta resulted in a synergistic effect on IL-1Ra mRNA and protein expression. Stimulation of freshly isolated mouse hepatocytes from male C57 BL/6 mice with HGF increased IL-1Ra mRNA and protein synthesis dose-dependently. A co-stimulation with HGF and IL-1beta had a synergistic effect on IL-1Ra mRNA expression but only a partially additive effect on IL-1Ra protein synthesis. HGF-induced IL-1Ra production was significantly decreased by the mitogen-activated protein kinase (MAPK) inhibitor PD98059. Accordingly, HGF stimulation specifically increased MAPK-dependent signalling pathway (p42/44). In contrast, in preactivated PBMC mRNA expression and protein synthesis of IL-1Ra, interleukin-10 (IL-10) and tumor necrosis factor-alpha (TNF-alpha) were unaffected after stimulation with HGF. In conclusion, our data suggest that HGF exerts anti-inflammatory effects by modulating the signal transduction cascade leading to increased expression of IL-1Ra, which might explain the protective and regenerative properties of this cytokine in animal models of liver failure.     

alpha3beta1 integrin induced suppression of the Caco-2 epithelial cell IL-1 signaling pathway leading to NF-(kappa)B activation

Intestinal epithelial cells (IECs) produce several potent cytokines in response to interleukin-1 (IL-1) and may play a role in the inflammatory response. Previously, we determined that treatment of the Caco-2 cells with a cross-linking anti-alpha3 integrin antibody resulted in a suppression of IL-1 induced cytokine secretion and mRNA levels, suggesting that the alpha3beta1 integrin may play a role in the regulation of IEC cytokine responses to IL-1. In this report, treatment of the Caco-2 cells with the anti-alpha3 integrin antibody resulted in a suppression of IL-1 induced levels of NF-kappaB binding activity in nuclear extracts, as determined by EMSA, as well as phosphorylation and degradation of the inhibitor, I(kappa)B(alpha). The anti-integrin antibody treatment was also found to suppress I(kappa)B kinase (IKK) activity and IKK(beta) phosphorylation. Culture of the Caco-2 cells on purified laminin-5, the ligand for the alpha3beta1 integrin, also resulted in suppression of IL-1 induced phosphorylation of I(kappa)B(alpha) and IKK(beta). Together with our previous findings, these results suggest that alpha3beta1 integrin binding results in a suppression of the IL-1 signaling pathway leading to the activation of NF-(kappa)B and ultimately IEC cytokine responses. These studies define a novel regulatory mechanism which may be important in the control of IEC cytokine responses during inflammation.     

Cytokine responses of human gingival fibroblasts to Actinobacillus actinomycetemcomitans cytolethal distending toxin

Actinobacillus actinomycetemcomitans is implicated in the pathogenesis of localized aggressive periodontitis, and has the capacity to express a cytolethal distending toxin (Cdt). Gingival fibroblasts (GF) are resident cells of the periodontium, which can express several osteolytic cytokines. The aims of this study were a) to investigate the role of Cdt in A. actinomycetemcomitans-induced expression of osteolytic cytokines and their cognate receptors in GF and b) to determine if the previously demonstrated induction of receptor activator of NFkappaB ligand (RANKL) by A. actinomycetemcomitans is mediated by these pro-inflammatory cytokines or by prostaglandin E(2) (PGE(2)). A. actinomycetemcomitans clearly induced interleukin (IL)-6, IL-1beta, and to a minimal extent, tumor necrosis factor (TNF)-alpha mRNA expression. At the protein level, IL-6 but not IL-1beta or TNF-alpha expression was stimulated. The mRNA expression of the different receptor subtypes recognizing IL-6, IL-1beta and TNF-alpha was not affected. A cdt-knockout strain of A. actinomycetemcomitans had similar effects on cytokine and cytokine receptor mRNA expression, compared to its parental wild-type strain. Purified Cdt stimulated IL-6, but not IL-1beta or TNF-alpha protein biosynthesis. Antibodies neutralizing IL-6, IL-1 or TNF-alpha, and the PGE(2) synthesis inhibitor indomethacin, did not affect A. actinomycetemcomitans-induced RANKL expression. In conclusion, a) A. actinomycetemcomitans induces IL-6 production in GF by a mechanism largely independent of its Cdt and b) A. actinomycetemcomitans-induced RANKL expression in GF occurs independently of IL-1, IL-6, TNF-alpha, or PGE(2).     

Proinflammatory cytokine IL-1 beta promotes tumor growth of Lewis lung carcinoma by induction of angiogenic factors: in vivo analysis of tumor-stromal interaction

Inflammatory conditions are associated with tumor development. IL-1beta is a multifunctional and proinflammatory cytokine that affects nearly all types of cells. To investigate the role of IL-1beta in tumor growth in vivo, we transduced the retroviral vector coding human IL-1beta gene into mouse Lewis lung carcinoma (LLC) cells and subsequently inoculated the transformant (LLC/IL-1beta) to syngeneic C57BL/6 mice. Tumors derived from LLC/IL-1beta grew faster (240%, day 18, vs null-vector control LLC/neo; p < 0.01) and showed more abundant vasculature (250%, vs LLC/neo; p < 0.05), whereas LLC/IL-1beta cells, LLC/neo cells, and wild-type LLC cells did not show any significant difference in the growth rate in vitro. As compared with LLC/neo cells, LLC/IL-1beta cells secreted 2-fold the amount of vascular endothelial growth factor and >10-fold the amount of macrophage-inflammatory protein-2 (CXCL2), one of whose main functions is angiogenesis. Although LLC/IL-1beta itself did not secrete hepatocyte growth factor (HGF), the tumor derived from LLC/IL-1beta cells also contained a >4-fold higher concentration of HGF, another angiogenic factor. In situ hybridization of HGF mRNA in LLC/IL-1beta tumor sections demonstrated that stromal fibroblasts and infiltrating cells overexpressed HGF mRNA. Moreover, when cultured in the presence of HGF in vitro, LLC/IL-1beta cells secreted even larger amounts of vascular endothelial growth factor and macrophage-inflammatory protein-2. The antiangiogenic agent TNP-470 and anti-CXCR2 Ab inhibited the tumor growth of LLC/IL-1beta cells in vivo. These results indicated that secreting IL-1beta into the tumor milieu induces several angiogenic factors from tumor and stromal cells and thus promotes tumor growth through hyperneovascularization.     

Toll-like receptor 9 mediates oral bacteria-induced IL-8 expression in gingival epithelial cells

Previously, we reported that various oral bacteria regulate interleukin (IL)-8 production differently in gingival epithelial cells. The aim of this study was to characterize the pattern recognition receptor(s) that mediate bacteria-induced IL-8 expression. Among ligands that mimic bacterial components, only a Toll-like receptor (TLR) 9 ligand enhanced IL-8 expression as determined by ELISA. Both normal and immortalized human gingival epithelial (HOK-16B) cells expressed TLR9 intracellularly and showed enhanced IL-8 expression in response to CpG-oligonucleotide. The ability of eight strains of four oral bacterial species to induce IL-8 expression in HOK-16B cells, and their invasion capacity were examined in the absence or presence of 2% human serum. The ability of purified bacterial DNA (bDNA) to induce IL-8 was also examined. Six out of eight strains increased IL-8 production in the absence of serum. Usage of an endosomal acidification blocker or a TLR9 antagonist inhibited the IL-8 induction by two potent strains. In the presence of serum, many strains lost the ability to induce IL-8 and presented substantially reduced invasion capacity. The IL-8-inducing ability of bacteria in the absence or presence of serum showed a strong positive correlation with their invasion index. The IL-8-inducing ability of bacteria in the absence of human serum was also correlated with the immunostimulatory activity of its bDNA. The observed immunostimulatory activity of the bDNA could not be linked to its CpG motif content. In conclusion, oral bacteria induce IL-8 in gingival epithelial cells through TLR9 and the IL-8-inducing ability depends on the invasive capacity and immunostimulating DNA.     

Contribution of IL-1 beta and TNF-alpha to the initiation of the peripheral lung response to atmospheric particulates (PM10)

Alveolar macrophages (AM) play a key role in clearing atmospheric particulates from the lung surface and stimulating epithelial cells to produce proinflammatory mediators. The present study examines the role of "acute response" cytokines TNF-alpha and IL-1 beta released by AM exposed to ambient particulate matter with a diameter of <10 microm (PM(10)) in amplifying the proinflammatory mediator expression by A549 cells and human bronchial epithelial cells (HBEC). The results showed that supernatants from human AM incubated 24 h with PM(10) (100 microg/ml) contained more TNF-alpha, IL-1 beta, granulocyte-macrophage colony stimulating factor, IL-6, and IL-8 than nonexposed AM supernatants. The 3-h treatment of A549 cells with PM(10)-exposed AM supernatants increased TNF-alpha, IL-1 beta, IL-8, regulated on activation normal T-cells expressed and secreted (RANTES), and leukemia inhibitory factor mRNA compared with the treatment with nonexposed AM supernatants and, compared with untreated A549 cells, additionally increased ICAM-1 and monocyte chemotactic protein-1 mRNA. Preincubating PM(10)-exposed AM supernatants with anti-IL-1 beta antibodies reduced all the above mediators as well as VEGF mRNA expression (P < 0.05), while anti-TNF-alpha antibodies were less effective (P > 0.05), and the combination of the two antibodies most effective. When HBEC were treated similarly, anti-TNF-alpha antibodies had the greatest effect. In A549 cells PM(10)-exposed AM supernatants increased NF-kappa B, activator protein (AP)-1 and specificity protein 1 binding, while anti-TNF-alpha and anti-IL-1 beta antibodies reduced NF-kappa B and AP-1 binding. We conclude that AM-derived TNF-alpha and IL-1 beta provide a major stimulus for the production of proinflammatory mediators by lung epithelial cells and that their relative importance may depend on the type of epithelial cell target.     

Regulation of rat pulmonary artery endothelial cell transforming growth factor-beta production by IL-1 beta and tumor necrosis factor-alpha.

Recent studies suggest that transforming growth factor-beta (TGF-beta) production is up-regulated at sites of tissue injury, inflammation and repair, or fibrosis. Endothelial cells represent a potentially important in vivo source of TGF-beta; however, the identity of endogenous modulators of TGF-beta production by these cells remains unclear. To address this issue, the effects of the cytokines, IL-1 beta, and TNF-alpha on TGF-beta production by rat pulmonary artery endothelial cells were examined. Conditioned media from cells treated with 0 to 20 ng/ml IL-1 beta and/or TNF-alpha were assayed for TGF-beta activity using a mink lung epithelial cell line. The results show that rat pulmonary artery endothelial cells secreted undetectable amounts of active TGF-beta in the absence of cytokines. However, upon acidification of the conditioned media before assay, a time-dependent increase in TGF-beta activity was noted in media from both untreated and cytokine-treated cells. However, both IL-1 beta and TNF-alpha treatment caused the secretion of significantly greater amounts of TGF-beta activity than control cells, in a dose-dependent manner, with maximal response obtained at cytokine doses of greater than 10 ng/ml. At equivalent doses of cytokine tested, the magnitude of the response was significantly greater with IL-1 beta. These responses were paralleled by increases in steady state mRNA levels for TGF-beta 1. Addition of both cytokines resulted in a synergistic response. Synergism with IL-1 beta was also noted with the fibrogenic agent bleomycin. Kinetic studies indicated that a minimum of 4 h of treatment with either IL-1 beta or TNF-alpha was required for detection of significant increases in either secreted TGF-beta activity or steady state TGF-beta 1 mRNA levels. Thus, endothelial cells could play a role in various TGF-beta-dependent processes in vivo, in situations wherein IL-1 beta and/or TNF-alpha may be present at comparable concentrations.